Design of a host specific molecule using molecular imprinting technique has been investigated since long time. Molecular imprinting methodology finds applications in bioseparation, enzyme mimics, chiral separations and antibody mimics. The technique creates selective binding sites in synthetic polymers (Mosbach, K., et al. TIBS, 19, 9–14, 1994).
The technique involves the polymerization of functional monomers in the presence of a template molecule. In the past various approaches such as covalent and non covalent interactions have been used to synthesize imprinted polymers (Kempe, M., Mosbach, K., Journal of Chromatography A, 694, 3–13, 1995). The template molecule binds to the active sites on the polymer via non covalent interactions such as ionic, hydrophobic or hydrogen bonding. Shea: K. J (TRIP, 5, 166–173, 1994) described the de novo synthesis of macromolecular binding and catalytic sites. Functional groups on macromolecular chains were bound non-covalently to polymerizable ligands which were then copolymerized with excess of cross linkers in the presence of macromolecular template.
Protein carbohydrate interactions are of low affinity. If relative density and spatial arrangement of ligands incorporated is optimized, then the binding between the substrates and the ligand can be substantially enhanced. The enhanced interactions are also desirable in affinity separations, drug delivery and biotechnology. Design of high affinity protein carbohydrate binding systems can provide an alternative strategy for the treatment of infectious diseases e.g. influenza and rotavirus. This has the advantage; as such agents will not result in pathogen resistance to antibiotics and drugs. A new approach to treat influenza is based on the principle of inhibition of virus binding on to the host cells. The inhibitors like sialic acid anchored to polymeric or liposomal carriers have been reported in the past.
Since monovalent interactions of natural oligosaccharides are weak, they need to be used in large quantities for an effective treatment. To overcome this problem polyvalent carbohydrate molecules can be synthesized (Zopf, D., Roth, S. Lancet 347, 1017, 1996). The concept of using polyvalent carbohydrate moieties is attractive since it provides a non-toxic therapeutic molecule to a wide range of human diseases. But synthesis of such compounds is critical and requires knowledge of the host-cell binding mechanism. So far molecular imprinting technique has been exploited for chiral separation. This involves interactions of polymerizable functional monomers around an imprinted molecule. Template molecule is then leached which leaves functional groups in the polymers at sites complementary to the template used during the synthesis. Shi H.; Tsai W. B.; Garrison M. D.; Ferrari S.; Ratner, B. D. (Nature, 398: 6728, 593–597, 1999) reported template-imprinted nanostructured surfaces for protein recognition. The investigators used radio-frequency glow-discharge plasma deposition to form polymeric thin films around proteins coated with disaccharide molecules. The disaccharides become covalently attached to the polymer film, creating polysaccharide-like environment around the template that exhibits highly selective recognition for the templated proteins, including albumin, immunoglobulin G, lysozyme, ribonuclease and “streptavidin. Direct imaging of template recognition is achieved by patterning a surface at the micrometer scale with imprinted regions.
Molecularly imprinted polymers can be used as specialty substrates for the separation of various biomolecules, The patent granted to our group (Vaidya; A. A,; Lele; B. S., Kulkarni; M. G; Mashelkar, R. A, U.S. Pat. No. 6,379,599, 2002) describes the process for preparation of molecularly imprinted polymers useful for separation of enzymes. The invention describes polymerization of complex comprising enzyme and affinity monomer, a comonomer and a crosslinker.
The molecularly imprinted polymers synthesized in the presence of biomolecules as templates impart the advantages of higher affinity and selectivity. Imprinted polymers in general display good recognition properties and are usually prepared in non-polar organic solvents such as chloroform or toluene. Biological recognition mainly occurs in hydrophilic environment and therefore it is important to synthesize MIPs containing ligands capable of interactions with a receptor molecule in the aqueous medium.
However, preparation of imprinted polymers in aqueous system has proven to be a difficult task, since the water molecule can destroy the hydrogen-bonding interactions between functional monomer and the template molecule. Moreover, commonly used cross-linkers do not dissolve in water.
Takeuchia, T.; Kugimiyaa A.; and Matsuia, J. (Materials Science and Engineering: C, 4: 4, 263–266, 1997) reported sialic acid-imprinted polymers using noncovalent interactions. Mosbach, Klaus; Mayes; Andrew G. (U.S. Pat. No. 5,959,050, 1999) reported molecularly imprinted polymer supports and their preparation via suspension polymerization. The suspension techniques according to the said invention provide molecularly imprinted polymers using a perfluorocarbon liquid containing polyoxyethylene ester groups as the dispersing phase. Most of the methods reported in the past utilize organic solvents for molecular imprinting.
Biomolecules such as enzymes and proteins are thermolabile and may undergo structural changes under the experimental conditions used for polymerization in the presence of these templates and lose their biological activity. Moreover, choice of such biomolecules as templates in organic solvents may alter their conformation and lead to loss of specificity.
Thus there is a necessity to synthesize imprinted polymers in which biomolecules will be solvent compatible, stable and hence can find wide range of applications such as biomolecular recoveries and medicine.
Site-specific interactions of ligand and the receptor are useful in immunoassays and biomolecule separations. The interacting molecules can be proteins or peptides, antibodies, enzymes, polysaccharides or glycoproteins that specifically bind to other substrate receptors in the suitable environment. A ligand so bound can be displaced from the binding site by altering environmental conditions.
Recent advancements in the field of glycoscience have demonstrated enhanced binding between carbohydrate ligands and specific receptors as a result of the polyvalency or cluster effect. Moreover, polyvalent materials also contribute to steric stabilization.
Literature highlights the advantages of polyvalent interactions and their application in medicine and biotechnology. The sialic acid moieties can be linked to polymer for the treatment of rotavirus (Mandeville, III, et al., U.S. Pat. No. 6,187,762, 2001). These moieties can inhibit or prevent rotavirus infection in mammals and humans.
Mammen, et al., (J. Med. Chem., 38, 4179–4190, 1995) reported polyacrylamides bearing pendent alpha sialoside groups as efficient inhibitors in agglutination of erythrocytes by influenza virus, suggesting the role of polyvalency. The affinity of the polyvalent inhibitor towards the surface of the virus is greatly enhanced compared to the monovalent sialic acid inhibitor. In addition high molecular weight polymers containing ligands inhibit binding between the virus and its receptor through steric exclusion.
Using controlled chemical synthesis methods such as molecular imprinting, it would be possible to control the spacing, steric accessibility, number of ligand molecules in the polymer. Moreover, molecular weight, density, solubility and physical structure of the imprinted polymeric conjugates can be manipulated as desired.
The synthesis of polyvalent copolymers by molecular imprinting technique can thus lead to unique advantages in various applications such as immunoassay, biomolecular recoveries and enhanced interactions.
Chitosan (Formula 4) is a linear, binary heteropolysaccharide consisting of 2-acetaamido-2-deoxy-β-D-glucose (GlcNAc; A-unit) and 2-amino-2-deoxy-D-glucose (GlcNAc, D-unit). The active site of lysozyme comprises subsites A–F. Specific binding of chitosan sequences to lysozyme begins with binding of the NAG Units in the subsite C. But, natural ligands derived from glucose are susceptible to microbial growth. Hence, there is a need to synthesize ligands similar to repeat units of chitosan, which will not be hydrolyzed by lysozyme. The polymers containing polyvalent NAG prepared using the imprinting technique reported here are expected to be more stable than chitin and chitosan.

Apart from the interacting ligand, its distribution in the polymer chain also plays a crucial role in influencing the efficiency of the inhibition. The synthesis of polymers bearing tailored molecular structure prepared by molecular imprinting could be the most effective method for enhancing substrate ligand interactions.
Synthesis of polyvalent carbohydrate ligands by the polymerization of the corresponding monovalent ligands in the presence of biomolecules comprising multiple binding sites, so as to enhance the binding between the polyvalent ligand and the imprint molecule in a subsequent stage has not been reported in the past to our knowledge. The imprinting methodology leads to enhanced interactions between the polyvalent ligands and the substrate than the polyvalent ligands containing identical moles of ligand synthesized in the absence of the template.
Many approaches have been reported in the past for controlled synthesis of amphiphilic block copolymers bearing pendent N-Acetyl-D-Glucosamine residues by living cationic polymerization. The interaction of the diblock copolymers with lectins was reported by Yamada et al. (Macromolecules, 32, 3553–3558, 1999). This methodology of synthesizing homopolymers and the block copolymers containing N-Acetyl-D-Glucosamine residues demonstrates significant increase in binding affinity for lectin. The applicability of the method is however limited by the need for very low temperature al}d stringent polymerization conditions Further the experimental conditions preclude the use of a protein molecule as a template during synthesis.
In our copending applications filed on the same date, “Oligomers and Preparation Thereof”—application Ser. No. 10/402,256 and “Macromer and preparation thereof”, application Ser. No. 10/812,838, we have claimed oligomers of NAG in which the NAG groups juxtaposed to one another, bind more effectively to lysozyme as reflected in values of binding constant (Kt,) and the inhibition concentrations (l50).
In the conventional technique of free radical copolymerization the distribution of monomers along the polymer chain depends upon the values of the monomer reactivity ratios, which are determined primarily by the intrinsic structure of the monomer. Consequently the distribution of the NAG units in the copolymers comprising monomers bearing NAG cannot be tailored at will using conventional copolymerization techniques. To overcome this problem we have devised a macromer synthesis method to ensure that the copolymers prepared using conventional free radical polymerization technique will always contain sequences of NAG units in juxtaposition. (Our co pending application Polymerizable Macromer and synthesis thereof—application Ser. No. 10/402,256)